T cells have the powerful capability to survey and destroy cells that have become infected or transformed. T cells express T cell receptors (TCRs), which specifically recognize short peptide fragments bound to major histocompatibility complex (MHC) molecules on the cell surface. Various studies have shown that greater infiltration of T cells (tumor infiltrating lymphocytes, TILs) in a patient's tumor correlates with improved prognosis. If a peptide/MHC is recognized with sufficient affinity by the TCR, the T cell is activated and releases granzymes and perforins that destroy the host-cell. Despite potential for the normal immune system to survey and destroy transformed cells, established cancers have clearly avoided such immune-mediated recognition and destruction. An overarching goal of immunotherapy is to promote TCR recognition of cancer pepMHC complexes so that the body's own immune system can be harnessed to destroy transformed tissues. During oncogenesis, cells express mutated and aberrantly overexpressed proteins via MHC molecules. One mechanism to achieve T cell recognition of cancerous tissue is to engineer high-affinity TCRs to bind to these cancer pepMHC complexes. Over 300 pepMHC complexes associated with different types of cancerous tissue have been identified, yet very few high-affinity TCRs specific for these pepMHC complexes have been isolated. The goal of this project is to engineer high-affinity TCRs against several cancer pepMHC complexes and test their ability to promote tumor regression in adoptive T cell models of cancer. Also, this project seeks to develop a more efficient method for isolating high-affinity TCRs. My specific aims are: Aim 1: To develop a high-affinity TCR against survivin/HLA-A2 using directed evolution and affinity maturation. Currently, we have isolated high-affinity TCRs against the cancer antigens Mart1 and WT1. Developing a high-affinity receptor against survivin will add to repertoire to receptors tested in other aims. Am 2: To test high-affinity TCRs in a chimeric antigen receptor (CAR) format that will mediate T cell activity against Mart1, WT1 and Survivin antigens in vivo. This aim will allow a better understanding of how high-affinity TCRs can be used in vivo, so that future clinical development is possible. Aim 3: To develop a method for rapid isolation of high-affinity TCRs using a universal scaffold. Current techniques for isolating high-affinity TCRs are time-consuming, involving isolation of T cell clones followed by one-by-one manipulation of the TCR genes. This aim will develop an optimal scaffold library so that rapid isolation of high-affinity TCRs can be achieved, facilitating their use in adoptive T cell immunotherapies for diverse cancers and many more patients.